Diversity and New Species of Ascomycota from Bamboo in China

Bamboo is an economically important crop that has gained prominence as an alternative to wood to reduce deforestation and ecosystem destruction. Diseases of bamboo that typically occur on leaves and stems can cause significant loss, reducing the quality and yield of the bamboo. However, there are few reports identifying the fungal species diversity and potential pathogens of bamboo. Here, we describe four new species of plant fungi from the leaves of bamboo within Fujian provinces, China. Fungi were isolated from diseased leaves collected within Fujian province and identified based on their morphological characteristics and multilocus phylogenies using nucleotide sequences derived from combined datasets of the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA gene (LSU), the large subunit of RNA polymerase I (rpb1), the translation elongation factor 1-α gene (tef1-α), and the partial beta-tubulin gene (tub2). These analyses helped reveal and clarify taxonomic relationships in the family Magnaporthaceae. The new species of bambusicolous fungi identified include two species of Bifusisporella, described as B. fujianensis sp. nov. and B. bambooensis sp. nov., and two species of Apiospora, described as A. fujianensis sp. nov. and A. fuzhouensis sp. nov. This study further expands the characterization and distribution of fungi associated with bamboo.


Introduction
The bamboo plant (Poales, Bambusoideae) family includes over 1400 different species of monocotyledon, mostly evergreen perennials.Bamboo encompasses the largest members of the grass family, occur naturally in a wide range of different ecosystems, and are cultivated as a highly versatile crop [1].Bamboos are well known to confer a number of beneficial ecological effects including carbon sequestration and erosion control, include some of the fastest growing plants known, have widespread ornamental use, and represent an important economic crop in regions where they are cultivated.Commercial applications of bamboo as a material for use in building/construction and fabrication of furniture, fabric, paper, cookware, cooking utensils, and many other items stems from its high strengthto-weight ratio and ease of cultivation that includes rapid plant growth.In addition, the plant is a food source for humans and other animals, notably giant and red pandas, as well as bamboo lemurs [2].Currently, approximately 80% of the world's bamboo species are found in the eastern and/or southern areas of Asia, with China having the richest bamboo resources in terms of highest diversity and overall cultivated area, accounting for more than 50% of the worldwide bamboo species [3,4].Bamboos plants show high resistance to microbial diseases, with fungal ascomycetes as the major microorganisms that limit the health and productivity of bamboo forests.Different bamboo fungi can infect various parts of the plant, resulting in nevus (gall-like "tumors"), spotted wilt, leaf damage/necrosis, and other symptoms and diseases that can lead to reduced quality and yield of the bamboo.Deleterious effects of Bambusicolous Ascomycota can impact economic development, and methods for the biological control of some bamboo fungi can reduce losses in bamboo forests and the cultivation industry, helping to maintain diversity, plant populations, and the varied beneficial ecological functions of bamboo forests [5].Over 1150 different species of ascomycetes may have some association (including pathogens, mutualists, and commensals) with bamboo, of which 350 asexual morphs, 240 hyphomycelia, and 110 coelomycetes have been tentatively identified [6].These fungi are mainly distributed in the Sordariomycetes, Dothideomycetes, and Eurotiomycetes, with the more representative families of bamboo fungi found in the Magnaporthaceae and Apiosporaceae families within the Sordariomycetes.
The Apiosporaceae fungal family belongs to the Ascomycota (Sordariomycetes Amphisphaeriales), with the type genus being Apiospora Sacc.introduced by Saccardo [16] and the type species being A. montagnei Sacc.The sexual morphology of Apiospora is characterized by hyaline ascospores surrounded by thick gelatinous sheaths [17][18][19].The asexual form of Apiospora is characterized by lenticular conidia that are spherical or subglobose and usually light brown to brown in color [20,21].Most Apiospora species are associated with plants as endophytes, with some species being economically important plant pathogens [22,23].
In this study, four fungal species, two of which represent new species, found growing on bamboo plants were identified and placed within the Magnaporthaceae, with their taxonomic placement determined based on morphological characteristics and molecular identification.The latter involved multilocus phylogenetic reconstructions using a combined dataset of the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA gene (LSU), the large subunit of RNA polymerase I (rpb1), and the translation elongation factor 1-α gene (tef1-α) nucleotide sequences.Similarly, four additional fungal isolates (again, with two representing new species) were identified and placed within the Apiosporaceae based upon morphological characteristics and molecular taxonomic and phylogenetic analyses using the combined marker loci sequence dataset (ITS + LSU + tef1-α + tub2).Our results identify two new species of Bifusisporella, Bifusisporella fujianensis sp.nov.and Bifusisporella bambooensis sp.nov.(Magnaporthaceae), and two new species of Apiospora, Apiospora fujianensis sp.nov., Apiospora fuzhouensis sp.nov.(Apiosporaceae), which are illustrated and described.This study expands the diversity of fungi infecting the economically and environmentally important bamboo plant.

Fungal Isolates and Morphology
Specimens were collected from diseased bamboo leaves in groves located in Fujian province, China.Tissue fragments with a total area of about 25 mm 2 were removed from the edges of the bamboo leaves in which disease spots, e.g., necrosis, wilting, and/or discoloration/blackening, were apparent.Samples were soaked in 75% ethanol for 45-60 s, then soaked in sterile deionized water for 45 s and washed with sterile water.Tissue fragments were then transferred to a 5% sodium hypochlorite solution for 30 s, followed by three washes in sterile deionized water for 60 s.The fragments were dried with sterilized filter paper and then transferred to the PDA plates which were incubated at 25 • C for 5-7 days following previously established procedures [24].Growing edges of fungal mycelia were transferred to new PDA plates and plates were incubated for 5-7 d.The procedure was continued until the fungal culture was pure (typically 2-4 times).To promote sporulation and observe the colony morphology, purified isolates were inoculated in the center of PDA and synthetic low-nutrient agar (SNA) plates and cultured at 25 • C under alternating conditions of 12 h near-ultraviolet light and 12 h dark [25].At 7 and 14 d of growth on PDA, photos of the colonies were taken with a digital camera, and the morphology of conidiomata, conidiophores, and conidiogenous cells was observed using a stereomicroscope (Nikon SMZ74, Tokyo, Japan).Samples were also prepared for analyses by scanning electron microscope (SEM, Nikon Ni-U; HITACHI SU3500) as described [26].Fungal micromorphology and structure were measured by Digimizer 5.4.4 software.Single colony purified cultures were cut and stored in 10% sterilized glycerin and sterile water at 4 • C for future detailed study.

Phylogenetic Analyses
Based on maximum likelihood (ML) and Bayesian inference (BI) analyses, phylogenetic trees were constructed to explore the phylogeny relationships of the fungal strains, grouping them into either the Magnaporthaceae or Apiosporaceae families.Corresponding gene loci of the reference sequences were downloaded from GenBank.Ophioceras dolichostomum (CBS 114926) was selected as an outgroup taxonomic unit for the phylogeny of Magnaporthaceae, and Sporocadus trimorphus (CBS 114203) was selected as an outgroup taxonomic unit for the phylogeny of Apiosporaceae.All sequences were aligned using the MAFFT v. 7 online service (http://mafft.cbrc.jp/alignment/server/,accessed on 2 February 2024) [36] and manually adjusted in BioEdit v.7.2.6.1 [37] and MEGA 7.0 [35].
In addition, four simultaneous Markov Chain Monte Carlo (MCMC) chains, starting with 2,000,000 generations of random trees, were sampled every 100th generation, resulting in a total of 20,000 trees.The first 25% of trees were discarded as burn-in of each analysis.Branches with significant Bayesian Posterior Probabilities (BYPP > 0.90) were estimated in the remaining 15,000 trees [38].Phylogenetic trees were plotted with FigTree v.1.4.4 [39] and embellished with Adobe Illustrator CS6.New sequences generated in this study have been deposited in GenBank (https://www.ncbi.nlm.nih.gov,accessed on 19 March 2024).

Phylogenetic Analyses
Samples of bamboo plants showing obvious fungal growth were collected from the Baizhu Garden of Fujian Agriculture and Forestry University and West Lake Park of Fuzhou City, Fujian Province, China.A total of eight fungal isolates with different morphological appearances were single-colony purified.For each fungal isolate, ~2637 bp of nucleotide sequences corresponding to portions of the ITS, LSU, rpb1, and tef1-αloci (ITS: 1-369; LSU: 370-1146; rpb1: 1147-1893; tef1-α: 1894-2775) were isolated.Based upon initial BLAST results, four of these sequences were isolated, combined with sequences from 68 closely related species as determined by BLAST searches, as well as homologous regions from Ophioceras dolichostomum (CBS 114926) and Ophioceras leptosporum (CBS 894.70) (Ophioceraceae, Magnaporthales) used as the outgroup for phylogenetic analyses.These analyses showed 1297 distinct patterns, with 1349 bp identical, 614 variable, including gaps, and 812 bp which were parsimony-informative.Maximum likelihood phylogenies were inferred using IQ-TREE under the TIM2 + R4 + F model for 5000 ultrafast bootstraps, as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test [nst = 6, rates = invgamma], with an average standard deviation of split frequencies = 0.005812.The topological results obtained from the ML analysis were consistent with the results of the BI analysis connecting the combined datasets.As a result, the ML tree is shown, and the BI posterior probabilities are placed on it (Figure 1).Based on phylogenetic resolution and morphological analysis (given below), we report two of the four isolates as new species of Magnaporthaceae: Bifusisporella fujianensis and Bifusisporella bambooensis.The new species B. fujianensis was most closely related to B. sichuanensis (SICAUCC 22-0073) (ML-BS: 96%, BYPP: 0.76), and B. bambooensis to B. sorghi (URM 7442, URM 7864) (ML-BS: 100%, BYPP: 1).
Initial BLAST results of sequences derived from the remaining four isolates indicated placement of these within the Apiosporaceae family.Analyses using sequences derived from the four genetic loci examined, namely the ITS + LSU + tef1-α + tub2 concatenated sequence dataset which had an aligned length of 1941 total characters (ITS: 1-507, LSU: 508-1341, tub2: 1342-1830, tef1-α: 1831-1941), supported the classification of two of the isolates as new species.Based on these and morphological data (below), a new species, Apiospora fujianensis, was identified, related to A. garethjonesii (SICAUCC 22-0028), with good support (ML-BS: 93% and BYPP: 0.98).Designation of the other new species, A. fuzhouensis, was similarly strongly supported (100% ML/1 PP), with the species forming a separate branch within Apiospora.For A. fuzhouensis loci analyses, 924 distinct patterns were identified, with 1186 bp constant, 157 variable and included gaps, and 598 bp which were parsimonyinformative. Maximum likelihood phylogenies were inferred using IQ-TREE [40], under the GTR + R3 + F model for 5000 ultrafast bootstraps [41], as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test [nst = 1, rates = invgamma] (Figure 2).
Culture characteristics: Colonies flattened on PDA, irregular black center, fading to white with white feathery margins, on SNA surface and reverse, white.Calculated growth rate was 1.0-1.4cm/day at 25 °C.The growth rate was 0.5 cm/day.Description: Leaf spots were pike-shaped, color gradually changing from blackish brown to white from outside to inside, Conidiomata bulging on agar, black to hyaline, aggregated and spherical, conidiophores reduced to conidiophores cells.Conidiogenous cells were phialidic, singly or in groups, curved, elongated, cylindrical, 7.  Culture characteristics: Colonies flattened on PDA, fluffy mycelium, black center with white margins over time; calculated growth rate of 1.3 cm/day at 25 °C.
Notes: In the present study, two strains were obtained from diseased leaves of bamboo and differed from each other with a high degree of statistical support (BYPP = 0.98 and ML-BS = 93%), although overall analyses indicated that both isolates represented different strains of the same species.
Apiospora fuzhouensis sp.nov.Z.Y.Zhao and J.Z. Qiu, (Figure 6).Notes: In the present study, two strains were obtained from diseased leaves of bamboo and differed from each other with a high degree of statistical support (BYPP = 0.98 and ML-BS = 93%), although overall analyses indicated that both isolates represented different strains of the same species.
Apiospora fuzhouensis sp.nov.Z.Y.Zhao and J.Z. Qiu, (Figure 6).Notes: Two strains were obtained from diseased leaves of bamboo and differed from each other with a high degree of statistical support (100% ML/1 PP, Figure 2), although overall analyses indicated that both isolates represented different strains of the same species.The nucleotide comparison of ITS sequences of A. garethjonesii (SICAUCC 22-0028) revealed 39 bp (39/542 bp, 7.2%) nucleotide differences.The nucleotide comparison of tub2 sequences of A. garethjonesii (SICAUCC 22-0028) revealed 20 bp (20/524 bp, 3.8%) nucleotide differences.Morphologically, the conidia of A. fuzhouensis were slightly smaller than those of A. garethjonesii (SICAUCC 22-0028).Therefore, the two strains are proposed as a new species.

Discussion
As interest in bamboo has intensified due to its wide range of beneficial environmental effects as well as agricultural, industrial, and even foodstuff uses, identification of pathogens, that can decrease quality and/or yield of the plant has also gained interest.Here, Notes: Two strains were obtained from diseased leaves of bamboo and differed from each other with a high degree of statistical support (100% ML/1 PP, Figure 2), although overall analyses indicated that both isolates represented different strains of the same species.The nucleotide comparison of ITS sequences of A. garethjonesii (SICAUCC 22-0028) revealed 39 bp (39/542 bp, 7.2%) nucleotide differences.The nucleotide comparison of tub2 sequences of A. garethjonesii (SICAUCC 22-0028) revealed 20 bp (20/524 bp, 3.8%) nucleotide differences.Morphologically, the conidia of A. fuzhouensis were slightly smaller than those of A. garethjonesii (SICAUCC 22-0028).Therefore, the two strains are proposed as a new species.

Discussion
As interest in bamboo has intensified due to its wide range of beneficial environmental effects as well as agricultural, industrial, and even foodstuff uses, identification of pathogens, that can decrease quality and/or yield of the plant has also gained interest.Here, we have identified four new species of fungi from diseased bamboo leaves found in Fujian Province, China.Identification was conducted using morphological and molecular phylogenetic analyses, with the former, i.e., characterization of the conidiomata, conidiophores, and conidiogenous cells used as important lines of evidence supporting species identification [42] and the latter (molecular approaches) allowing for phylogenetic placement and confirmation of new species designations.
Two of new species were identified as belonging to the Bifusisporella genus.Previously, Silva et al. isolated an endophyte, Bifusisporella sorgh, from healthy sorghum leaves in Brazil [15], and another endophyte, B. sichuanensis, has been reported from leaves of Sichuan poplar [43].Most Bifusisporella species have sickle-shaped ditype conidia and are commonly found in Poaceae.The newly described species in this report, B. fujianensis, grouped with B. sichuanensis, but was distinct from the latter in both morphology and multilocus sequence analyses, whereas B. bambooensis potentially represents a separate clade.Morphological differences between the species were evident, particularly concerning the conidia (Table 3).The remaining two new species identified in this report were found to belong to the Apiospora genus.Crous and Groenewald synonymized Apiospora with Arthrinium [44]; however, with additional genetic data from the Arthrinium type species, A. caricicola, Apiospora and Arthrinium were separated into two distinct genera [19].Biogeographically, most specimen of Arthrinium have been found in temperate and boreal zones, whereas those of Apiospora have been mainly collected in tropical and subtropical regions, with the latter genus displaying a relatively wider distribution area.Currently, therefore, based on the molecular phylogenetic analysis of multigene loci (ITS, LSU, and exon sequences of tef1-α and tub2), Arthrinium and Apiospora are considered to represent independent lineages within the Apiosporaceae [19], confirming that the overall genetic, morphological, and ecological differences between Apiospora and Arthrinium are sufficient to support the taxonomic separation of the two genera.Apiospora are characterized by round/lenticular conidia, which are mainly found in Poaceae.Based on morphology and molecular analyses, Apiospora fujianensis sp.nov.and Apiospora fuzhouensis sp.nov.were described as two new species within Apiospora.
As a neo-tropical region, fungal species diversity in Fujian and surrounding areas appears particularly robust [26,27].However, thus far, only a few species of fungi have been found in bamboo leaves, with our understanding of the diversity of fungal parasitism on bamboo incomplete.This is likely due to bamboo being particularly hardy and resis-

Figure 1 .
Figure 1.ML tree generated from combined ITS, LSU, rpb1, and tef1-α sequence data of Magnaporthaceae and Pyriculariaceae.The maximum likelihood (ML) bootstrap support values and Bayesian posterior probabilities (BYPP) bootstrap support values above 70% and 0.90 are shown at the first and second position.Species with sequences obtained in this study are in boldface and newly generated sequences were indicated in red.Ophioceras dolichostomum (CBS 114926) and O.leptosporum (CBS894.70)(Ophioceraceae) were used as the outgroup.Yellow-green strips represent different neighboring species.

Figure 1 .
Figure 1.ML tree generated from combined ITS, LSU, rpb1, and tef1-α sequence data of Magnaporthaceae and Pyriculariaceae.The maximum likelihood (ML) bootstrap support values and Bayesian posterior probabilities (BYPP) bootstrap support values above 70% and 0.90 are shown at the first and second position.Species with sequences obtained in this study are in boldface and newly generated sequences were indicated in red.Ophioceras dolichostomum (CBS 114926) and O.leptosporum (CBS894.70)(Ophioceraceae) were used as the outgroup.Yellow-green strips represent different neighboring species.

Figure 2 .
Figure 2. Phylogram of Apiospora based on combined ITS, LSU, tef1-α, and tub2 genes.ML bootstrap support values (ML-BS ≥ 70%) and Bayesian posterior probability (BYPP ≥ 0.90) are shown as first and second position above nodes, respectively.Strains from this study are shown in red.Some branches were shortened according to the indicated multipliers.

Figure 2 .
Figure 2. Phylogram of Apiospora based on combined ITS, LSU, tef1-α, and tub2 genes.ML bootstrap support values (ML-BS ≥ 70%) and Bayesian posterior probability (BYPP ≥ 0.90) are shown as first and second position above nodes, respectively.Strains from this study are shown in red.Some branches were shortened according to the indicated multipliers.

Table 1 .
The primer sequences and programs in this study.

Table 2 .
Species names, voucher or culture codes, hosts or substrate, locations, and corresponding GenBank accession numbers of DNA sequences used in this study.
Notes: newly generated sequences are in bold.

Table 3 .
The location, hosts or substrate, and main morphological characters of Bifusisporella.